Abstract The tumor microenvironment severely limits the efficacy of various immunotherapies. For adoptive T cell therapy, the need to select/engineer and expand T cells with targeted antigen specificity while still preserving their effector function and homing capacities poses additional challenges. We and others have demonstrated that Stat3, a key oncogenic transcription factor constitutively activated in diverse cancer cells and important for tumor cell survival, is also activated in tumor stromal immune cells and potently immunosuppressive. Our preliminary studies show that targeted Stat3 gene ablation in myeloid cells results in tumor DC activation, tumor Treg cell reduction and heavy infiltration of CD8+ T cells in tumors, leading to effective antitumor immune responses. Furthermore, in mice with a Stat3-/- myeloid compartment, adoptively transferred CD8+ T cells proliferate well and are highly activated in the tumor draining lymph nodes. We were also able to capture live images by multi-photon confocal microscopy of transferred Stat3-/- CD8+ T cells efficiently infiltrating tumors, where they proliferate, leading to activation of tumor antigen-specific immune responses. We have begun to understand the cellular and molecular mechanisms that allow Stat3 in both tumor myeloid cells and T cells to impede T-cell mediated antitumor immunity. In this application, we propose to further define these cellular and molecular mechanisms and thereby identifying additional targets to optimize Stat3 targeting and T cell therapy. To facilitate potential future clinical translation, we will also test two reagents, both of which have shown positive effects on the tumor immunologic environment, for their potential to improve T cell therapies. One is a novel Toll-like receptor (TLR) agonist-siRNA conjugate, a technology platform we have recently developed, and is capable of immune activation and targeted gene silencing of desired immunologic checkpoints such as Stat3 in myeloid and B cells. The other is an FDA-approved tyrosine kinase inhibitor, sunitinib, as our preliminary data suggested it inhibited Stat3 in tumor cells, tumor-infiltrating dendritic cells and myeloid-derived suppressor cells. Additionally, we will test antitumor efficacy by silencing Stat3 in T cells ex vivo with lentiviral-siRNAs. The proposed studies may lead to new strategies to expand and activate transferred T cells in vivo, thereby overcoming several major hurdles facing T cell therapy, and significantly improve its efficacy for cancer treatment.